Estrogen synthesis is catalyzed by aromatase P450 (product of hCYP19 gene), which is expressed at very high levels in human placenta. hCYP19 gene expression is markedly induced during differentiation of human placental cells in culture; trophoblast differentiation and induction of hCYP19 expression are blocked by hypoxia. We recently observed that estrogen-related receptor 3 (ERR3) is O2-regulated and plays a crucial role together with estrogen receptor 1 (ER1) in the induction of hCYP19 expression during human trophoblast differentiation. Notably, ERR3 regulates K+ homeostasis in heart, stomach and kidney of mice via control of a number of hypertension-associated genes; further, ERR3 single nucleotide polymorphisms are correlated with altered blood pressure in humans. Based on the potential importance of placentally-derived estrogens in vascular adaptations during pregnancy and dysregulation of vascular remodeling in preeclampsia, a major goal of our research is to further define genetic and epigenetic mechanisms in O2-dependent regulation of hCYP19 gene expression during human trophoblast differentiation. In light of the suggested role of placental hypoxia in the pathogenesis of preeclampsia, we will identify ERR3 target genes that control Na+/K+ homeostasis and vascular tone in human and mouse placenta. In Specific Aim 1, cultured human trophoblasts will be used to investigate genetic and epigenetic mechanisms whereby critical transcription factors and coregulators upregulate hCYP19 expression during human trophoblast differentiation and block its expression with hypoxia. In Specific Aim 2, we will test the hypothesis that O2-dependent human trophoblast differentiation and induction of hCYP19 expression are mediated, in part, by specific microRNAs (miRNAs/miRs) that regulate expression of key transcription factors and coregulators. In Specific Aim 3 we will investigate roles of ERR3 and of ER1 in O2-mediated regulation of placental genes that modulate K+ and blood pressure homeostasis and analyze their expression in placentas from preeclamptic vs. normal pregnancies and from wild-type and ERR3 null mice. We anticipate that our studies will shed new light on the etiology of this devastating disease.